Non-thrombogenic semipermeable membrane and method for making same

ABSTRACT

The invention concerns a non-thrombogenic semipermeable membrane comprising a support semipermeable membrane and an anticoagulant suitable for treating blood and plasma by extracorpreal circulation. The invention is characterized in that: the support membrane is made essentially of a polyacrylinitrile, bearing anionic groups or groups capable of being anionic, the surface of the support semipermeable membrane designed to be in contact with blood or plasma is coated successively with a cationic polymer and an anticoagulant. The invention also concern an exchanger for treating blood or plasma by extracorporeal circulation comprising two sections separated by a non-thrombogenic semipermeable, membrane and a method for making the exchanger.

[0001] The present invention concerns the field of blood and plasmatreatment by extracorporeal circulation, in particular by dialysis,haemofiltration and plasmapheresis. It relates to a compositesemipermeable membrane comprising a semipermeable support membranecoated with an anticoagulant agent, which reduces the thrombogeniccharacter of the support.

[0002] The present invention also relates to exchangers for treatingblood or plasma by extracorporeal circulation, comprising this compositesemipermeable membrane, as well as to methods for manufacturing theseexchangers.

[0003] Throughout this text, the term “semipermeable membrane” is usedto denote a flat semipermeable membrane or a bundle of semipermeablehollow fibres. Also, throughout this text, the term “exchanger” is usedto denote an exchanger for treating blood or plasma by extracorporealcirculation, which generally comprises two compartments separated by asemipermeable membrane, each provided with two accesses, a firstcompartment being intended for the circulation of the patient's blood orplasma, and a second compartment being intended for the circulation ofused liquid.

[0004] The treatment of a uraemic patient's blood or plasma requires theuse of an extracorporeal blood circuit connected to an exchanger. Theblood, in the extracorporeal circuit, is subjected to a major thromboticrisk which it is necessary to prevent by effective anticoagulation. Thisis why, even though the materials used for the manufacture of exchangersare selected to be as biocomoatible as possible so that, in particular,coagulation reactions do not take place or take place at a relativelybenign levels, an anticoagulant agent, generally heparin (fractionatedor non-fractionated) is customarily injected into the patient's blood.

[0005] The amount of heparin injected into the patient conventionallyvaries from 7500 to 11,000 international units (IU) per treatmentsession, or more in certain cases.

[0006] In common practice, the anticoagulation of blood rarely presentsdifficulties. In certain patients, however, heparin can causeundesirable secondary reactions, in particular haemorrhaging. Otherside-effects reported in the literature are thrombocytopoenia (drop inthe number of platelets) allergies, osteopoross (observed in the eventof prolonged heparin adrinistration), increases in transaminases(hepatic enzymes) and hyperlipidaemia. Sensitivity to heparin isvariable from one patient to another and as a function of time for agiven patient.

[0007] For patients with a high risk of haemorrhaging, there is a methodof extracorporeal blood treatment without an anticoagulant, whichconsists in preventing thrombosis of the blood in the extracorporealcirculation circuit and in the dialyser by frequently rinsing thecircuit and the dialyser with physiological saline in order to flushtherefrom the thrombi being formed. This method involves high bloodcirculation rates, which are not always tolerated by patients.

[0008] Furthermore, this treatment without an anticoagulant, which istechnically difficult, requires specific training of the nursing staff,rigorous compliance with the operating protocol, strict monitoring ofthe dialysis in order to prevent incidents, and early detection ofincidents so that they can be dealt with.

[0009] With a view to avoiding the injection of anticoagulant into theextracorporeal blood circuit, a great deal of research work has beenundertaken in order to improve the haemocompatibility of semipermeablemembranes by binding heparin to the surface of the membrane intended tobe placed in contact with the blood, by means of ionic or covalentbonding (see J. Pelissié “Héparinisation de surface” [surfaceheparinization] in RMB (1994) 16, 7, 290-291).

[0010] The techniques described for the ionic binding of heparin aim tocreate quaternary amine groups on the surface of the semipermeablemembrane to be treated. Heparin, which is negatively charged owing toits sulphate and carboxylic groups, can react with quaternary aminegroups. However, rapid release of heparin due to the weakness of theintermolecular bonding has been observed, which presents the drawback ofreducing the antithrombogenic character of the surfaces intended to bein contact with the blood and of promoting the appearance of undesirableside-effects due to freed heparin. In order to compensate for thecontinuous release of heparin, large amounts of this anticoagulant mustbe bound to the membrane, which makes it difficult to control theanticoagulation of the patient's blood and increases the cost.

[0011] The techniques described for covalent binding involve specificand complex chemical reactions allowing heparin to become attached tothe surfaces of the materials intended to be placed in contact with theblood. These techniques, however, since they cause a substantialmodification of the chemical nature of the materials, are not generallysuitable for semipermeable membranes because they would risk provoking asignificant reduction in the fundamental properties of these membranes,such as diffusive and convective transport capacities and the capacityfor adsorbing undesirable substances. There is furthermore a risk ofreducing the antithrombogenicity by inactivation of heparin.

[0012] The only exchanger marketed to date comprising a semipermeablemembrane treated with an anticoagulant agent in order to reduce itsthrombogenic character is the haemofilter with the brand name DURAFLOfrom the company BAXTER. This semipermeable membrane is based onpolysulphone.

[0013] Certain semipermeable membranes consisting essentially of acopolymer of acrylonitrile and at least one anionic or anionizablemonomer, which is o'efinically unsaturated, such as the copolymermanufactured by HOSPAL under the brand name AN69, are acknowledged to bemuch less thrombogenic than cellulosic membranes. The implementation ofdialysis without heparin using these membranes requires, however, highblood circulation rates [at least 400 ml/min according to the article“Heparin-free hemodialysis with a polyacrylonitrile membrane”, by RobertH. Barth et al., Vol. XXXV Trans Am Soc Artif Intern Organs (1989)],which is not always tolerated by all patients.

[0014] It is a first object of the present invention to reduce thethrombogenic character of semipermeable membranes consisting essentiallyof a copolymer of acrylonitrile and at least one anionic and anionizablemonomer by binding an anticoagulant agent to these membranes, whilekeeping the other fundamental properties of these membranes at anacceptable level, that is to say their capacity for diffusive andconvective transport (water permeability, selective permeability tomolecules with a weight-average molecular weight less than that ofalbumin), their capacity for adsorbing undesirable substances and theirmechanical properties.

[0015] It is a second object of the present invention to bind, stably,to the surface of semipermeable membranes consisting essentially of acopolymer of acrylonitrile and at least one anionic and anionizablemonomer, an anticoagulant agent which can exert its anticoagulantactivity without being eluted and released rapidly into the blood orplasma during the treatment by extracorporeal circulation.

[0016] It is a third object of the present invention to reduce theamount of anticoagulant agent injected into the patient during anextracorporeal blood treatment session carried out by means of anexchanger provided with a semipermeable membrane consisting essentiallyof a copolymer of acrylonitrile and at least one anionic and anionizablemonomer, to which an anticoagulant agent is bound.

[0017] In order to achieve these objects, the present invention providesa composite semipermeable membrane comprising a semipermeable supportmembrane and an anticoagulant agent suitable for treating blood andplasma by extracorporeal circulation, characterized in that:

[0018] the semipermeable support membrane consists essentially of apolyacrylonitrile carrying anionic or anionizable groups;

[0019] the surface of the semipermeable support membrane intended to beplaced in contact with the blood or plasma is coated successively:

[0020] with a cationic polymer carrying cationic groups which arecapable of forming an ionic bond with the anionic or anionizable groupsof the polyacrylonitrile, the cationic polymer comprising chains whosesize is sufficient for them not to pass through the semipermeablesupport membrane; and

[0021] with an anticoagulant agent carrying anionic groups which arecapable of forming an ionic bond with the cationic groups of the saidcationic polymer.

[0022] In certain cases, the present invention furthermore makes itpossible to reduce the total amount of the bound or unboundanticoagulant agent needed for implementing an extracorporeal bloodtreatment session by means of an exchanger provided with a semipermeablemembrane consisting essentially of a copolymer of acrylonitrile and atleast one anionic and anionizable monomer, to which an anticoagulantagent is bound, and hence to reduce the cost of the treatment sessionand the undesirable side-effects of the anticoagulant agent.

[0023] According to the invention, the chains of the cationic polymerhave a sufficiently large steric hindrance for them not to pass throughthe semipermeable support membrane, which makes it possible for thesechains to become bound essentially to the surface of the membrane byionic bonding. The amount of cationic polymer needed for treating thesemipermeable support membrane is consequently moderate since a bulktreatment of the semipermeable support membrane with penetration of thecationic polymer into the pores is not required.

[0024] Preferably, the cationic polymer is prepared by ultrafiltrationusing a semipermeable membrane which is identical to the semipermeablesupport membrane or which has a cut-off threshold equal to or greaterthan that of the semipermeable support membrane, in order to exclude thecationic polymer chains capable of passing through the semipermeablesupport membrane.

[0025] The composite semipermeable membrane according to the inventionhas three major advantages:

[0026] firstly, the binding of the anticoagulant agent by ionic bondingtakes place almost only at the surface of the semipermeable membrane.The bound anticoagulant agent, which is accessible to the coagulationproteins, can consequently exert an effective anticoagulant activityduring a session of blood and plasma treatment by extracorporealcirculation.

[0027] secondly, the coagulation time (KCT, for activated cephalin time)of blood or plasma purified with the aid of the composite semipermeablemembrane is fairly close, or equivalent, to that of normal blood orplasma not supplemented with an anticoagulant agent.

[0028] thirdly, the thrombogenic character of the compositesemipermeable membrane is very greatly reduced when comparing it withthat of the semipermeable support membrane which it contains.

[0029] According to one variant of the invention, the cationic polymeris a polyamine which is preferably hydrophilic and carries primary,secondly, tertiary or quaternary amine groups. The cationic polymer ispreferably a polyethyleneimine (PEI). The amount of PEI deposited andbound can vary between approximately 1 mg and approximately 30 mg per m²of membrane (including the end points).

[0030] The anticoagulant agent suitable for the invention must not betoxic.

[0031] According to another variant of the invention, the anticoagulantagent carrying anionic groups belongs to the family of glycoaminoglycanshaving an anticoagulant activity. Preferably, this agent essentiallyconsists of heparin (fractionated or non- fractionated). The amount ofheparin deposited and bound can vary between approximately 200 IU andapproximately 20,000 IU per m² of membrane (Including the end points),preferably between approximately 500 IU and approximately 10,000 IU perm² of membrane (including the end points). The amount of heparin boundis selected according to the type of treatment for which the exchangeris intended:

[0032] intermittent treatment (session of from 3 to 6 hours) forpatients suffering from chronic or acute renal insufficiency orcontinuous treatment (12 to 96 hours) for patients suffering from acuterenal insufficiency;

[0033] treatment during which no anticoagulant agent is injected intothe patient's vascular circuit.

[0034] The amount of heparin bound is less than the amount of heparinwhich is injected into the patient during a conventional treatment(which as of the order of from 7500 to 11,000 IU or more in the eventthat the membrane is rinsed with a heparin solution before the blood iscirculated in the extracorporeal circuit).

[0035] The invention is suitable for the preparation of compositesemipermeable membranes from a semipermeable support membrane consistingessentially of a polyacrylonitrile carrying anionic or anionizablegroups which are selected from sulphonic, phosphonic, carboxylic,sulphuric, phosphoric groups and from the corresponding salified groups.

[0036] Preferably, the anionic or anionizable groups of thepolyacrylonitrile are acid sulphonic groups or salified sulphonicgroups.

[0037] Advantageously, the semipermeable support membrane consistsessentially of a copolymer of acrylonitrile and sodium methallylsulphonate, such as the copolymer with the brand name AN69 manufacturedby HOSPAL, with which the best performance has been achieved.

[0038] The invention also relates to an exchanger for treating blood orplasma by extracorporeal circulation, comprising two compartmentsseparated by a semipermeable membrane having a surface oriented towardsa first compartment intended for the circulation of blood or plasma,characterized in that that the surface of the semipermeable membraneoriented towards the first compartment is coated successively with acationic polymer and with an anionic anticoagulant agent.

[0039] The composite semipermeable membrane may be in the form of a flatmembrane or a bundle of hollow fibres.

[0040] Preferably, the exchanger which comprises the compositesemipermeable membrane according to the invention is sterilized andready for use. It hence does not require any special handling by itsuser.

[0041] The present invention also relates to a method for reducing thethrombogenic character of an exchanger for treating blood or plasma byextracorporeal circulation, comprising two compartments separated by asemipermeable membrane having a surface oriented towards a firstcompartment intended for the circulation of blood or plasma, comprisingthe following successive stages:

[0042] (a) preparing a semipermeable membrane, In the form of a flatmembrane or a bundle of hollow fibre, from a solution ofpolyacrylonitrile carrying anionic or anionizable groups;

[0043] (b) assembling the various components of the exchanger, inparticular fitting the semipermeable membrane or a bundle of hollowfibres in a case;

[0044] (c) preparing a solution containing at least one cationic polymercarrying cationic groups which are capable of forming an ionic bond withthe anionic or anionizable groups of the polyacrylonitrile, the cationicpolymer comprising only polymer chains whose size is sufficient for themnot to pass through the semipermeable membrane, and bringing thissolution into contact with the surface of the semipermeable membraneintended to be placed in contact with the blood, it being possible tocarry out stage (c) before or after stage (b); when the semipermeablemembrane is flat, the cationic polymer solution may be sprayed onto thesurface of the membrane;

[0045] (d) in the event that stage (c) is carried out subsequently tostage (b), purging the exchanger of the solution containing the cationicpolymer;

[0046] (e) preparing a solution containing, in the dissolved state, atleast one anticoagulant agent carrying anionic groups which are capableof forming an ionic bond with the cationic groups of the said cationicpolymer, and bringing this solution into contact with the surface of thesemipermeable membrane intended to be placed in contact with the blood,stage

[0047] (e) being implemented after stage (c) but before or after stage(b);

[0048] (f) in the event that stage (e) is carried out subsequently tostage (b), purging the exchanger of the solution containing theanticoagulant agent.

[0049] In the event that the cationic polymer is PEI, the aforementionedstage (c) may be carried out according to the following conditions:

[0050] PEI concentration: from 0.04 to 20 g/L

[0051] medium: water; glycerinated water; saline buffers; salinesolutions

[0052] pH: from 5 to 12

[0053] treatment rates (in the case of treating the membrane bycirculation in the instrument): from 50 to 500 mL/min

[0054] duration: from 1 to 30 minutes

[0055] open circuit or closed circuit

[0056] under these conditions, the surface PEI concentration is between1 and 30 mgr.

[0057] In the event that the anticoagulant agent is heparin, theaforementioned stage (e) may be carried out according to the followingconditions:

[0058] heparin concentration: from 1 to 100 TU/L

[0059] medium: water; glycerinated water; saline buffers; salinesolution

[0060] pH: from 5 to 10

[0061] treatment rates (in the case of treating the membrane bycirculation in the instrument):from 50 to 500 mL/min

[0062] duration: from 1 to 30 minutes

[0063] open circuit or closed circuit

[0064] under these conditions, the surface heparin concentration isbetween 200 and 20,000 IU/m², preferably between 500 and 10,000 IU/m².

[0065] Optionally, the flat membrane or the bundle of hollow fibres isglycerinated at the end of stage (a), whence the need to de-glycerinatebefore undertaking the aforementioned stage (c).

[0066] Optionally, the semipermeable membrane is rinsed in order toremove the excess bound cationic polymer, either after stage (c) whenstage (c) is carried out before stage (b), or after stage (d).

[0067] Optionally, the semipermeable membrane is rinsed in order toremove the excess unbound anticoagulant agent, either after stage (e)when stage (e) is carried out before stage (b), or after stage If).

[0068] Optionally, the semipermeable membrane is re- glycerinated,depending on the case, at the end of stage (e) or (f), after theoptional rinsing stages.

[0069] In the scope of the invention, the sterilization of theexchanger, without a significant effect on the composite semipermeablemembrane, may be sterilization by irradiation, in particular by gammairradiation, or sterilization with ethylene oxide. The sterilization ofthe exchanger may be implemented at two specific times during the methodof manufacturing the exchanger.

[0070] According to a first variant, the exchanger is sterilized whenthe semipermeable membrane based on polyacrylonitrile carrying anionicor anionizable groups is coated with the said cationic polymer, then thetreatment using the solution containing at least one anticoagulant agentis performed extemporaneously.

[0071] According to a second variant, the exchanger is sterilized whenthe semipermeable membrane based on polyacrylonitrile carrying anionicor anionizable groups is coated with the said cationic polymer and thesaid anticoagulant agent.

[0072] Other characteristics and advantages of the invention will becomeapparent on reading the following examples. Reference will also be madeto the appended drawings and figures, in which:

[0073]FIG. 1 represents the procedures for preparing a cationic polymer,such as polyethyleneimine (PEI), by ultrafiltration;

[0074]FIG. 2 represents the change in the PEI concentration in theultrafiltrate;

[0075]FIG. 3 represents the distribution of the weight-average molecularweights of the non-fractionated PEI, denoted PEI P, (LUPASOL P, fromBASF) and fractionated PEI;

[0076]FIG. 4 represents the change in the coagulation time (KCT) ofsheep blood subjected to an extracorporeal circulation test using adialyser with a flat AN69 membrane treated with fractionated PEI,gamma-sterilized, then treated extemporaneously with heparin;

[0077]FIG. 5 represents the change in the coagulation time (KCT) ofsheep blood subjected to an extracorporeal circulation test using adialyser with hollow AN69 fibres treated with fractionated PEI, thenheparinated, and finally gamma-sterilized before use.

[0078] The assaying methods used to evaluate the semipermeable membraneswhich have been described are as follows:

[0079] Treatment of the Blood Samples Prior to the Assays

[0080] The blood sampled, when leaving the dialysers, during thedialysis sessions of the following examples is immediately placed in thepresence of sodium citrate which, by chelation of the calcium ions,stops any coagulation activity. The sample is then centrifuged at roomtemperature at 3000 revolutions per minute for 15 minutes. Thesupernatant plasma is collected and stored at −20° C. until the assay.

[0081] Determination of the Coagulation Time (KCT, for Kaolin-CephalinTime)

[0082] The KCT determination is carried out using the assay kit marketedunder the name C.K. PREST® by the company DIAGNOSTICA STAGO.

[0083] The KCT makes it possible to assess the lengthening of thecoagulation time of a citrated plasma due to the deficit of certaincoagulation factors or the presence of an anticoagulant such as heparin.In the latter case, the lengthening of the coagulation time isproportional to the amount of heparin present. The KCT determinationhence makes it possible to assess the anticoagulation level of theblood. The method of measuring this coagulation time (expressed inseconds) is known, and it is carried out after recalcification andaddition of an activator.

[0084] Heparin Assay in Non-Plasmatic Medium

[0085] Heparin is determined in simple media (water and electrolytes) byspectrophotometry after formation of a complex with azure A (maximumabsorption at 630 nm).

[0086] Test of Extracorporeal Circulation on a Sheep:

[0087] The dialysers tested in the examples are rinsed with aphysiological saline solution (heparinated or non-heparinated)circulated in the blood compartment (2 L at 200 mL/min.). The dialysatecompartment is filled by ultrafiltration. Depending on the case, thesheep blood is anticoagulated by injecting heparin into the jugular veinfive minutes before the start of the session. The extracorporeal bloodcirculation is carried out at the rate of 200 mL/min. using a HOSPALBSM22 pump (carotid access and jugular return). The input and outputpressures are recorded. The circulation is stopped when the bloodpressure exceeds 500 mmHg at the entry of the dialyser, giving evidence15 of coagulation in the circuit.

EXAMPLE 1

[0088] This example describes a method of preparing a cationic polymer,in this case a polyethyleneimine WE (PEI), aiming to remove byfractionation the smallest polymer chains (with little steric hindrance)capable of penetrating into the pores of the semipermeable membrane tobe treated, and passing through it.

[0089]FIG. 1 illustrates the procedures for preparation of the PEI,which comprises the following stages:

[0090] a—preparing, in a tank 1, a solution of 1.5 liters of a solutionof PEI with a weight-average molecular weight of 750 k Dalton (LUPASOLP, from BASF) and 50 g per liter, in distilled water;

[0091] b—circulating, in a closed circuit, the solution in the bloodcompartment of a dialyser 2 with hollow fibres (brand name FILTRAL 16,manufactured by the company HOSPAL INDUSTRIE, France), equipped with amembrane (working area of 1.6 m²) made of AN69 35 (copolymer ofacrylonitrile and sodium methallyl sulphonate), at the raze of 300 mlper minute;

[0092] c—simultaneously with stage b, ultrafiltration at the rate of 60ml per minute with addition of water into the tank 1, at the same rate.

[0093] The duration of the preparation is 136 minutes.

[0094] The assay of the PEI present in the ultrafiltrate is determinedin water by spectrophotometry after formation of a coloured complex withcobalt II thiocyanate (maximum absorption at 304 nm).

[0095] The change in the PEI concentration in the ultrafiltrate is givenon appended FIG. 2.

[0096] Under the conditions of the aforementioned method, the amount ofPEI removed by ultrafiltration is 32 g, which represents 43% of theoriginal PEI.

[0097] The distribution by molecular weight (Mw) of the non-fractionatedPEI (denoted PEI P) and the fractionated PEI is determined by a stericexclusion chromatograph (ultrahydrogel column from the company WATERS)and is represented on appended FIG. 3. FIG. 3 shows that the molar massof the smallest chains of fractionated PEI is of the order of 10,000g/mole.

EXAMPLES 2 Reference Example 2a

[0098] A dialyser with a flat membrane made of AN69 (brand name CRYSTAL4000, manufactured by HOSPAL INDUSTRIE, France), having a working areaof 1.5 m² and sterilized by gamma irradiation, is rinsed by circulating,in the blood compartment, 2 liters of physiological saline containing5000 IU of non-fractionated heparin.

[0099] An extracorporeal blood circuit comprising the dialyser is thenconnected to the vascular circuit of a sheep. No anticoagulant isinjected into the sheep's blood.

[0100] On FIG. 4, curve 1 gives the chance in the KCT during the 30minutes of the blood circulation. Coagulation took place at the end of30 minutes.

Example 2b According to the Invention

[0101] A dialyser was manufactured according to the invention by thecompany HOSPAL INDUSTRIE (France). One face of a flat membrane made ofAN69, having a working area of 1.5 m², is treated by sprayingfractionated PEI (see Example 1) at the concentration of 5 g/kg into a40/60 by weight water/glycerol mixture. The amount of fractionated PEIdeposited is 10 mg/m².

[0102] This membrane is fitted in a dialyser so that the treated face isoriented towards the blood compartment of the dialyser.

[0103] The dialyser is then sterilized by gamma irradiation (36 K Gy).

[0104] Just before its use, the sterilized dialyser is rinsed using asolution of physiological saline and non-fractionated heparin, asindicated in Reference Example 2a.

[0105] An extracorporeal blood circuit comprising the dialyser is thenconnected to the vascular circuit of a sheep. No anticoagulant isinjected into the sheep's blood.

[0106] It was possible to circulate the sheep's blood for 6 hours in theextracorporeal circuit without coagulation taking place (the terminationafter 6 hours is voluntary and does not correspond to coagulation in thecircuit). Curve 2 of FIG. 4 gives the KCT level throughout thecirculation. The KCT remains at a normal level, signifying that heparinis not released, whereas a KCT of more than 100 seconds is customarilynecessary for correct operation of a blood treatment in anextracorporeal circulation circuit.

EXAMPLES 3 Reference Example 3a

[0107] A dialyser (brand name FILTRAL 20, manufactured by HOSPALINDUSTRIE, France), equipped with a bundle of hollow AN69 fibres with aworking area of 2 m², sterilized with ethylene oxide, is rinsed bycirculating, in the blood compartment, 2 liters of physiological salinecontaining 10,000 IU of non-fractionated heparin.

[0108] An extracorporeal blood circuit comprising the dialyser is thenconnected to the vascular circuit of a sheep, after 5000 IU ofnon-fractionated heparin have previously been injected into the sheep.

[0109] The coagulation of blood in the extracorporeal circuit took placeafter the blood had circulated therein for 2 hours.

[0110] The change in the KCT level is represented on FIG. 5, by curve 1.

Example 3b According to the Invention

[0111] A dialyser (brand name NEPHRAL 300, manufactured by HOSPALINDUSTRIE, France), equipped with a bundle of hollow AN69 fibres with aworking area of 1.3 m², is treated by circulating, in the bloodcompartment, a solution of fractionated PEI prepared as in Example 1 (1g/L in water, closed circuit on 200 mL, 5 minutes, 200 mL/min.). Thisdialyser undergoes a second treatment by circulating, in the samecompartment, a solution of non-fractionated heparin (5 IU/mL in aphosphate solution (Na2HPO₄ at 10 mM) closed circuit on 3 L, 200mL/min., 5 or 30 min.). The dialyser is then purged with air (0.3 barfor 30 s) Under these conditions, the amount of PEI bound is of 2 theorder of 15 mg/m², the amount of heparin bound is 2500 and 6800 IU/m²(measured according to the method of assaying heparin in a non-plasmaticmedium).

[0112] This dialyser is then sterilized by gamma as irradiation. justbefore the dialyser is used, 2 liters of physiological saline arecirculated in the blood compartment in order to rinse it. Anextracorporeal blood circuit comprising the dialyser is then connectedto the vascular circuit of a sheep. No anticoagulant is injected intothe sheep's blood.

[0113] It was possible to maintain the circulation of blood in theextracorporeal circuit without injecting heparin into the extracorporealcircuit for 3 and 6 hours without coagulation making place, and curves 2and 3 of FIG. 5 indicate that the KCT levels remained normal.

EXAMPLES 4

[0114] Examples 4 correspond to series of clinical trials carried outwith dialysers according to the present invention and dialysers notaccording to the present invention.

Comparative Examples 4a and 4b

[0115] 4a) Untreated dialysers of brand name NEPHRAL 300 are used in aseries of clinical trials carried out under the following conditions:

[0116] 6 patients;

[0117] 108 haemodialysis sessions lasting 4 hours with addition ofheparin;

[0118] prior to each haemodialysis session, the dialyser is rinsed bycirculating 1 liter of a physiological saline solution containing 5000IU of heparin in the blood compartment of the dialyser;

[0119] at the end of each haemodialysis session, the blood is returnedto the patient by rinsing the blood compartment of the dialyser with 1liter of physiological saline.

[0120] 4b) Dialysers of brand name NEPHRAL 300 were treated successivelyin the following way:

[0121] circulating 1 liter of water, with a flow rate of 200 ml/min, inthe blood compartment of the dialyser in order to remove the glycerinepresent in the hollow fibres;

[0122] circulating 1 liter of a solution of non- fractionated PEI with aweight-average molecular weight of 750 K Dalton (LUPASOL P from BASF),in the blood compartment of the dialyser;

[0123] purging with air;

[0124] sterilizing by gamma irradiation;

[0125] prior to each haemodialysis session, the dialyser is rinsed bycirculating 1 liter of a physiological saline solution containing 5000IU of heparin in the blood compartment of the dialyser;

[0126] at the end of each haemodialysis session, the blood is returnedto the patient by rinsing the blood compartment of the dialyser with 1liter of physiological saline.

[0127] A series of 27 clinical trial sessions is carried out with thesedialysers.

[0128] Results of the Series of Clinical Trials (4a) and (4b)

[0129] After returning the blood, the dialysers are evaluated visually,on a scale of 1 to 4:

[0130] score 1 (poor): the colour of the entire dialyser is red andindicates significant coagulation of the blood in the semipermeablemembrane;

[0131] score 2 (medium): the colour of approximately half the dialyseris red;

[0132] score 3 (good): only a few red traces in the dialyser;

[0133] score 4 (excellent): the dialyser is not red. Example 4a Example4b (*) Average value over all 6200 8400 the sessions of the (min: 3000)(min: 4500) amount of heparin added, (max: 8000) (max: 12000) withoutincluding the amount of heparin bound at the time of rinsing (min value,max value) (**) Average value over all 3.0 1.9 the sessions of the (min:2) (min: 1) visual assessment of the (max: 4) (max: 3) dialysers (minvalue, max value)

[0134] Conclusion

[0135] The treatment with a non-fractionated PEI leads to significantconsumption (adsorption) of heparin. Furthermore, the absorbed heparinis not active since the dialysers thus treated in Examples (4b) lead tocoagulation problems.

Examples 4c According to the Present Invention

[0136] A flat membrane made of AN69 is treated by spraying fractionatedPEI, prepared under the conditions described above in Example 1, in aproportion of 10 mg/m² of membrane.

[0137] Dialysers of the CRYSTAL type are fitted with this membrane (1.25m² working surface of membrane per dialyser) and are sterilized by gammairradiation.

[0138] A series of clinical trials is carried out with these dialysersunder the following conditions:

[0139] 13 patients;

[0140] 256 haemodialysis sessions;

[0141] prior to each haemodlalysis session, the dialyser is rinsed bycirculating 2 liters of a physiological saline solution containing 5000IU of heparin per liter in the blood compartment of the dialyser;

[0142] haemodialysis sessions lasting 4 hours with or without additionof heparin (see table);

[0143] at the end of each haemodialysis session, the blood is returnedto the patient by rinsing the blood compartment of the dialyser with 1liter of physiological saline. Average value over all 6000 5000 40003000 2000 1000 0 Examples (4c) of the amount of heparin added, withoutincluding the amount of heparin bound at the time of rinsing (*) Averagevalue over all 4 (39) 4 (39) 4 (38) 4 (35) 4 (31) 4 (28) 4 (28) thesessions of the 2 (1)  2 (2)  2 (4)  2 (4)  2 (4)  visual assessment ofthe 1 (1)  1 (1)  1 (1)  dialysers (number of sessions)

[0144] Conclusion

[0145] The treatment with a fractionated PEI, followed by a treatmentwith heparin, makes it possible to carry out dialysis with lower amountsof anticoagulant injected into the patient.

[0146] Comments

[0147] In comparative Examples (4a) and (4b), the KCT is kept between 90and 120 s.

[0148] In Examples (4c), it is the injected heparin dose which is fixed(between 0 and 6000) and, in all cases, the KCT at the end of a sessionis less than 50, which is a considerable advantage for the patient (nomore heparin circulating at the end of dialysis, and therefore no morerisk of haemorrhaging).

[0149] After return of the blood, the dialysers of this series ofclinical trials are evaluated as previously under the conditionsdescribed above for Examples (4a) and (4b).

1. Composite semipermeable membrane comprising a semipermeable supportmembrane and an anticoagulant agent suitable for treating blood andplasma by extracorporeal circulation, characterized in that: thesemipermeable support membrane consists essentially of apolyacrylonitrile carrying anionic or anionizable groups; the surface ofthe semipermeable support membrane intended to be placed in contact withthe blood or plasma is coated successively: with a cationic polymercarrying cationic groups which are capable of forming an ionic bond withthe anionic or anionizable groups of the polyacrylonitrile, the cationicpolymer comprising chains whose size is sufficient for them not to passthrough the semipermeable support membrane; and with an anticoagulantagent carrying anionic groups which are capable of forming an ionic bondwith the cationic groups of the said cationic polymer.
 2. Membraneaccording to claim 1, characterized in that the anionic or anionizablegroups of the polyacrylonitrile are selected from sulphonic, phosphonic,carboxylic, sulphuric, phosphoric groups and from salts of theaforementioned groups.
 3. Membrane according to claim 1, characterizedin that the anionic or anionizable groups of the polyacrylonitrile areacid sulphonic groups or salified sulphonic groups.
 4. Membraneaccording to claim 3, characterized in that the semipermeable supportmembrane consists essentially of a copolymer of acrylonitrile and sodiummethallyl sulphonate.
 5. Membrane according to one of claims 1 to 4,characterized in that the cationic polymer is a polyamine.
 6. Membraneaccording to claim 5, characterized in that the cationic polymer is apolyethyleneimine.
 7. Membrane according to claim 6, characterized inthat the amount of polyethyleneimine deposited is between approximately1 mg and approximately 30 mg per m² of membrane (including the endpoints).
 8. Membrane according to one of claims 1 to 7, characterized inthat the cationic polymer is prepared by ultrafiltration using asemipermeable membrane which is identical to the semipermeable supportmembrane or which has a cut-off threshold equal to or greater than thatof the semipermeable support membrane, in order to exclude the cationicpolymer chains capable of passing through the semipermeable supportmembrane.
 9. Membrane according to one of claims 1 to 8, characterizedin that the anticoagulant agent carrying anionic groups belongs to thefamily of glycoaminoglycans having an anticoagulant activity. 10.Membrane according to claim 9, characterized in that the anticoagulantagent essentially consists of heparin.
 11. Membrane according to claim10, characterized in that the amount of anticoagulant agent deposited isbetween approximately 200 IU and approximately 20,000 IU per m² ofmembrane (including the end points).
 12. Exchanger for treating blood orplasma by extracorporeal circulation, comprising two compartmentsseparated by a semipermeable membrane having a surface oriented towardsa first compartment intended for the circulation of blood or plasma,characterized in that the semipermeable membrane is a compositesemipermeable membrane according to one of claims 1 to 11, and in thatthe surface of the membrane oriented towards the first compartment iscoated successively with a cationic polymer and with an anionicanticoagulant agent.
 13. Exchanger according to claim 12, characterizedin that the composite semipermeable membrane is a flat membrane. 14.Exchanger according to claim 12, characterized in that the compositesemipermeable membrane consists of a bundle of hollow fibres.
 15. Methodfor reducing the thrombogenic character an exchanger for treating bloodor plasma by extracorporeal circulation, comprising two compartmentsseparated by a semipermeable membrane having a surface oriented towardsa first compartment intended for the circulation of blood or plasma, themethod comprising the following successive stages: (a) preparing asemipermeable membrane, in the form of a flat membrane or a bundle ofhollow fibres, from a solution of polyacrylonitrile carrying anionic oranionizable groups; (b) assembling the various components of theexchanger, in particular fitting the semipermeable membrane or a bundleof hollow fibres in a case; (c) preparing a solution containing at leastone cationic polymer carrying cationic groups which are capable offorming an ionic bond with the anionic or anionizable groups of thepolyacrylonitrile, the cationic polymer comprising only polymer chainswhose size is sufficient for them not to pass through the semipermeablemembrane, and bringing this solution into contact with the surface ofthe semipermeable membrane intended to be paced in contact with theblood or plasma, it being possible to carry out stage (c) before orafter stage (b); (d) in the event that stage (c) is carried outsubsequently to stage (b), purging the exchanger of the solutioncontaining the cationic polymer; (e) preparing a solution containing, inthe dissolved state, at least one anticoagulant agent carrying anionicgroups which are capable of forming an ionic bond with the cationicgroups of the said cationic polymer, and bringing this solution intocontact with She surface of the semipermeable membrane intended to beplaced in contact with the blood, stage (e) being implemented afterstage (c) but before or after stage (b); (f) in the event that stage (e)is carried out subsequently to stage (b), purging the exchanger of thesolution containing the anticoagulant agent.
 16. Method according toclaim 15, characterized in that the semipermeable membrane is rinsed inorder to remove the excess unbound cationic polymer, either after stage(c) when stage (c) is carried out before stage (b), or after stage (d).17. Method according to claim 15 of 16, characterized in that thesemipermeable membrane is rinsed in order to remove the excess unboundanticoagulant agent, either after stage (e) when stage (e) is carriedout before stage (b), or after stage (f).
 18. Method according to claim15, 16 or 17, characterized in that the exchanger is sterilized when thesemipermeable membrane based on polyacrylonitrile carrying anionic oranionizable groups is coated with the said cationic polymer, then thetreatment using the solution containing at least one anticoagulant agentis performed extemporaneously.
 19. Method according to claim 15, 16 or17, characterized in that the exchanger is sterilized when thesemipermeable membrane based on polyacrylonitrile carrying anionic oranionizable groups is coated with the said cationic polymer and the saidanticoagulant agent.
 20. Method according to claim 15, characterized inthat the cationic polymer is prepared by ultrafiltration using asemipermeable membrane which is identical to the semipermeable supportmembrane or which has a cut-off threshold equal to or greater than thatof the semipermeable support membrane, in order to exclude the cationicpolymer chains capable of passing through the semipermeable supportmembrane.